The objective of this proposal is to gain an understanding of the control mechanisms operative in the regulation of eukaryotic gene expression, using adenovirus type 5 (Ad5) early region 2A as the model system. The gene product of early region 2A is a 72,000 dalton single-stranded DNA-binding protein (DBP) that is involved in regulating viral gene expression in at least four ways: (1) the initiation and elongation steps of DNA synthesis, (2) the regulation of transcription of several of the Ad5 early genes, (3) stability of early mRNAs, and (4) the host-range of the virus. The immediate goal of this work is to precisely define within the DBP domains that are involved in each of the functions of the protein. To accomplish this, we will generate mutations in the DBP by site-specific mutagenesis techniques and analyze the mutant DBPs for their ability to bind to single-stranded DNA (specific aim #1), and to help adeno-associated virus replicate in human cells (specific aim #2). In-depth probing of the physical properties of the protein will be performed if we can isolate a cell line capable of complementing adenovirus mutants defective for growth because of altered DBPs, or if expression of mutated DBPs can be obtained in prokaryotic systems. The isolation of the complementing cell line would allow a thorough in vivo analysis of the functions of mutant DBPs (specific aim #3). The successful outcome of these experiments will provide significant insight into the molecular mechanisms by which eukarotic gene expression is regulated.